Benchwork
Plan
- Miniprep pTXB1His and send for sequencing
- Start more cultures of new colonies for more minipreps
- Don't forget to miniprep pTXB1 (unmodified) as well
- Streak expression strain (ER2566) on LB so competent cells and a glycerol stock can be made
Experiments
- Glycerol stock
- 750μL pTXB1His/DH10B QC colony #1 culture from yesterday + 250 μL 60% glycerol (sterile) → tube #79
- → -80°C
- Miniprep of pTXB1His
- follow Miniprep vacuum protocol for pTXB1His/DH10B QC colony #1 culture from yesterday
- Quantify [pTXB1His miniprep #1]
- 95μL H2O
- + 5μL miniprepped DNA from step 2
- → measure absorbance at 260 nm and 280 nm
- DNA sequencing
- Miniprep cultures of pTXB1His and pTXB1 in DH10B
- 10mL LB + 10μL 100 mg/mL Amp + pTXB1His/DH10B QC colony #2 (see Results below)
- 10mL LB + 10μL 100 mg/mL Amp + pTXB1His/DH10B QC colony #3 (see Results below)
- 10mL LB + 10μL 100 mg/mL Amp + pTXB1/DH10B colony (from last week)
Results
Transformations
- Four plates yesterday:
- 200μL experimental transformation from yesterday: 1 colony
- → pTXB1His/DH10B QC colony #2
- 200μL control transformation from yesterday: 0
- 800μL experimental transformation re-plated from 2 days ago: 1 colony
- → pTXB1His/DH10B QC colony #3
- 800μL control transformation re-plated from 2 days ago: 0
Miniprep
[math]\displaystyle{ [pTXB1His\#1]=(0.114AU+0.012AU)\times50\frac{\frac{\mu g}{mL}}{AU}\times\frac{100\mu L}{5\mu L}=102\frac{\mu g}{mL} }[/math]
[math]\displaystyle{ \frac{A_{260}}{A_{280}}=\frac{0.114AU+0.012AU}{0.067AU+0.011AU}=1.82 }[/math]
Sequencing
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link to alignment
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